Harry A. Peters

The antigen levels of components of the urokinase-type plasminogen activator (uPA) system of plasminogen activation are correlated with prognosis in several types of cancers, including breast cancer. In the present study involving 2780 patients with primary invasive breast cancer, we have evaluated the prognostic importance of the four major components of the uPA system [uPA, the receptor uPAR (CD87), and the inhibitors PAI-1 and PAI-2]. The antigen levels were determined by ELISA in cytosols prepared from primary breast tumors. The levels of the four factors significantly correlated with each other; the Spearman rank correlation coefficients (r(s)) ranged from 0.32 (between PAI-2 and PAI-1 or uPAR) to 0.59 (between uPA and PAI-1). The median duration of follow-up of patients still alive was 88 months. In the multivariate analyses for relapse-free survival (RFS) and overall survival (OS), we defined a basic model including age, menopausal status, tumor size and grade, lymph node status, adjuvant therapy, and steroid hormone receptor status. uPA, uPAR, PAI-1, and PAI-2 were considered as categorical variables, each with two cut points that were established by isotonic regression analysis. Compared with tumors with low levels, those with intermediate and high levels showed a relative hazard rate (RHR) and 95% confidence interval (95% CI) of 1.22 (1.02-1.45) and 1.69 (1.39-2.05) for uPA, and 1.32 (1.14-1.54) and 2.17 (1.74-2.70) for PAI-1, respectively, in multivariate analysis for RFS in all patients. Compared with tumors with high PAI-2 levels, those with intermediate and low levels showed a poor RFS with a RHR (95% CI) of 1.30 (1.14-1.48) and 1.76 (1.38-2.24), respectively. Similar results were obtained in the multivariate analysis for OS in all patients. Furthermore, uPA and PAI-1 were independent predictive factors of a poor RFS and OS in node-negative and node-positive patients. PAI-2 also added to the multivariate models for RFS in node-negative and node-positive patients, and in the analysis for OS in node-negative patients. uPAR did not further contribute to any of the multivariate models. A prognostic score was calculated based on the estimates from the final multivariate model for RFS. Using this score, the difference between the highest and lowest 10% risk groups was 66% in the analysis for RFS at 10 years and 61% in the analysis for OS. Moreover, separate prognostic scores were calculated for node-negative and node-positive patients. In the 10% highest risk groups, the proportion of disease-free patients was only 27 +/- 6% and 9 +/- 3% at 10 years for node-negative and node-positive patients, respectively. These proportions were 86 +/- 4% and 61 +/- 6% for the corresponding 10% lowest risk groups of relapse. We conclude that several components of the uPA system are potential predictors of RFS and OS in patients with primary invasive breast cancer. Knowledge of these factors could be helpful to assess the individual risk of patients, to select various types of adjuvant treatment and to identify patients who may benefit from targeted therapies that are currently being developed.

Vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been reported to be associated with a poor prognosis in primary breast cancer and in several other cancer types. In the present study, we have measured with ELISA the levels of VEGF in cytosolic extracts of 845 primary breast tumors of patients who developed a recurrence during follow-up. All of the patients received tamoxifen (n = 618) or cyclophosphamide, methotrexate, 5-fluorouracil (CMF) or 5-fluorouracil, Adriamycin, cyclophosphamide (FAC) chemotherapy (n = 227) as first-line systemic therapy after diagnosis of advanced disease. VEGF levels were not related to age or menopausal status but were negatively related to the cytosolic levels of estrogen receptor and progesterone receptor (P < 0.0001). In patients who relapsed within 1 year after primary surgery, tumor VEGF levels were higher than in patients who showed a longer disease-free interval (P = 0.0005). In patients with a first relapse in the viscera, VEGF levels were higher compared with those that relapsed to the bone or soft tissue (P = 0.0004). In univariate analysis for response to first-line tamoxifen therapy, patients with high or intermediate levels showed a poor rate of response, compared with patients with low tumor-VEGF levels (P = 0.0001). Similarly, in multivariate analysis for response to tamoxifen treatment, corrected for age, site of relapse, disease-free interval, and estrogen receptor and progesterone receptor status, VEGF status was an independent predictive factor (P = 0.009). In concordance, higher levels of VEGF were associated with a short progression-free survival and postrelapse overall survival (both, P < 0.0001). On first-line chemotherapy, the rate of response decreased with higher tumor levels of VEGF, both in univariate (P = 0.003) and in multivariate analysis (P = 0.004). Furthermore, higher VEGF levels were associated with a short progression-free survival (P = 0.003) and postrelapse overall survival (P = 0.001). In conclusion, the tumor VEGF level is an important independent marker that predicts a poor efficacy of both tamoxifen and chemotherapy in advanced breast cancer. Knowledge of the tumor level of VEGF might be helpful in selecting individual patients who may benefit from treatments with antiangiogenic agents combined with conventionally used drugs.

Urokinase-type plasminogen activator (uPA) may be responsible for the invasive and metastasizing capacity of tumor cells. Evidence has been presented that primary breast cancer patients with tumors containing high levels of uPA experience a worse prognosis. In the present study we have assessed uPA status in routinely prepared cytosols of 671 primary human breast tumors and have evaluated its association with disease-free and overall survival. Isotonic regression analysis with length of disease-free survival as an end point revealed 1.15 ng/mg protein as the best cutoff point to discriminate between uPA positive (32% of the tumors) and uPA negative. In both Cox univariate and multivariate regression analysis (including also patient's age, menopausal status, lymph node status, and the number of positive lymph nodes, tumor size, and estrogen and progesterone receptor status), uPA positivity was significantly associated with increased rates of relapse and death. Corrected for all relevant factors in multivariate analyses for subgroups of patients, uPA positivity was significantly associated with an increased relapse rate in the subgroups of node-negative (P = 0.002; relative failure rate, 2.33), node-positive (P < 0.0001; relative failure rate, 1.95), postmenopausal (P < 0.0001; relative failure rate, 2.59), and steroid receptor-positive patients (P < 0.0001, relative failure rate, 2.76). We conclude that uPA positivity of human primary breast tumors is an important independent variable for the identification of patients at high risk for recurrence, also in clinically important subgroups of patients.

PURPOSE: Evaluation of the relationship between plasminogen activator inhibitor-1 (PAI-1) and the metastatic potential of primary breast cancer, and to compare the prognostic impact of PAI-1 in multivariate analysis with those of conventional prognostic factors, including steroid-hormone receptors, and those of urokinase plasminogen activator (uPA), pS2-protein (PS2), and cathepsin D. PATIENTS AND METHODS: Cell biologic prognostic factors were analyzed in 657 cytosols routinely prepared from frozen-tissue biopsies that were submitted to our laboratory for the assessment of steroid-hormone receptor status. The median duration of follow-up in patients still alive at the time of analysis was 48 months. Estrogen receptor (ER) and progesterone receptor (PgR) status were assessed by radioligand binding assay, PS2, and cathepsin D by radiometric immunoassay, and uPA and PAI-1 by enzyme-linked immunosorbent assay (ELISA). RESULTS: PAI-1 levels were found to be strongly positively correlated with the rates of relapse (P < .0001) and death (P < .001). Relating the levels of PAI-1 with those of other cytosolic prognostic factors, we found a positive association with the metastasis-related proteases uPA (P < .0001) and cathepsin D (P < .0001). On the other hand, PAI-1 levels were found to be negatively correlated with ER (P < .005) and PgR (P < .001), and the estrogen-regulated pS2-protein (P < .001), which are proteins associated with a favorable prognosis. In multivariate regression analysis for 5-year relapse-free survival, and using an optimized cutoff point for discrimination between PAI-1-positive and -negative, independent predictors of the rate of relapse were found to be PAI-1 (P < .0001) and uPA (P = .01) of the cytosolic parameters, and tumor size, lymph node status, and premenopausal age of the clinical parameters. In multivariate analysis in patients with node-negative disease, only PAI-1 (P < .001) and tumor size (P = .03) were positively and premenopausal age negatively (P < .001) associated with the rate of relapse. In patients with node-positive disease, PAI-1 (P < .001), uPA (P = .02), tumor size (P < .001), and the number of positive lymph nodes (P < .001) were all positively associated with the rate of relapse. CONCLUSION: We conclude that the PAI-1 level measured in routinely prepared cytosols is an important parameter to predict the metastatic potential in both node-negative and node-positive human primary breast cancer.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay is widely used for in vitro measurement of the metabolic viability of cell cultures subjected to different culture conditions. This convenient assay, which is based on the ability of viable cells to produce formazan, can be affected significantly by a number of conditions. These conditions can be roughly divided into groups, firstly influences which affect the spectrum of the produced formazan and secondly influences which affect the amount of formazan produced per cell. We studied the various chemical and biochemical aspects involved in the MTT assay. Our data indicated that microscopical viewing of cell cultures before and after performing the assay, a medium renewal with a well-defined MTT-incubation medium at the end of the culture period and regular cell counting are essential steps to ensure a reliable performance of the MTT assay. In conclusion, providing the necessary precautions are taken, the MTT assay can be used reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival.